The long term goal of the proposed research is to understand, in detail, the role of spliceosomal snRNAs in catalysis of the splicing reaction. In the active spliceosome, U6 and U2 snRNAs form a base-paired complex that helps position the reactants of the first step of splicing for catalysis. Previously published work and preliminary studies show that an in vitro-assembled, base paired U6/U2 complex resembling the one forming in vivo can catalyze a reaction closely related to the first step of splicing. This finding will be pursued under three specific aims. 1) Characterization of the splicing-related catalytic activity of U6 and U2 snRNAs. To conclusively demonstrate that the reaction catalyzed by the in vitro-assembled U6/U2 complex is indeed identical to the first step of splicing, the chemistry of the reaction will be directly analyzed by nuclease digestion of site-specifically labeled products, followed by TLC analysis. In addition, by manipulating the structure of the U6/U2 complex, the basis for the selection of the scissile phosphate will be defined. 2) Determination of the structural organization of the catalytic U6/U2 complex. In vivo data has indicated the importance of the three-dimensional structure of the U6/U2 complex in spliceosomal catalysis. Using a battery of chemical probing reagents and crosslinking assays, the structural architecture of the in vitro- assembled U6/U2 and its interactions with the splicing substrates will be defined. 3) Functional analysis of the sequence elements required for catalysis. Mutational analysis of the snRNAs in the spliceosome has revealed a functionally crucial role for a number of nucleobases and backbone phosphates. The molecular basis of the function of these required elements will be defined using nucleotide analog interference mapping (NAIM). Using a similar approach, the functional significance of abundant post-transcriptional modifications in the U6 and U2 snRNAs will be defined. Almost all human pre-messenger RNAs undergo multiple splicing events, and alternative splicing is not only one of the most important means of regulation of gene expression, it is also largely responsible for generating proteomic diversity in eukaryotes. Disturbances in the pattern of pre-mRNA splicing have been linked to a broad spectrum of human diseases ranging from genetic and neurodegenerative diseases to malignancies.